PARP-1 Activation Requires Local Unfolding of an Autoinhibitory Domain.

Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.

Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage.

Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity.

Genetics and Genomics in Clinical Practice: The Views of Wisconsin Physicians.

INTRODUCTION: Decreasing costs and increased availability of genetic testing and genome sequencing mean many physicians will consider using these services over the next few years. Despite this promising future, some argue the present roadmap for translating genetics and genomics into routine clinical practice is unclear. OBJECTIVE: We conducted a pilot study to explore Wisconsin physicians' views, practices and educational desires regarding genetic and genomic testing. METHODS: Our study consists of an Internet survey (n=155) conducted in August and September 2015 and follow-up phone interviews with a portion of survey participants. Physicians of all specialties were invited to participate. Variables measured include physicians' general knowledge and experience regarding genetic and genomic testing, attitudes and perceptions toward these tests, testing intentions, and educational desires. Sociodemographic variables included gender, age, and medical specialty. RESULTS: In our exploratory survey of Wisconsin physicians, adult primary care providers (PCPs) lagged behind other providers in terms of familiarity and experience with genetic and genomic testing. PCPs in our sample were less likely than other physicians to feel their training in genetics and genomics is adequate. Physicians younger than 50 were more likely than older colleagues to feel their training is adequate. CONCLUSIONS: Our exploratory study suggests a gap in physician education and understanding regarding genomic testing, which is fast becoming part of personalized medical care. Future studies with larger samples should examine ways for physicians to close this gap, with special focus on the needs of PCPs.

Yawning as a behavioral marker of mild motion sickness and sopite syndrome.

INTRODUCTION: Severe motion sickness is easily identifiable with sufferers showing obvious behavioral signs, including emesis (vomiting). Mild motion sickness and sopite syndrome lack such clear and objective behavioral markers. We postulate that yawning may have the potential to be used in operational settings as such a marker. This study assesses the utility of yawning as a behavioral marker for the identification of soporific effects by investigating the association between yawning and mild motion sickness/sopite syndrome in a controlled environment. METHODS: Using a randomized motion-counterbalanced design, we collected yawning and motion sickness data from 39 healthy individuals (34 men and 5 women, ages 27-59 yr) in static and motion conditions. Each individual participated in two 1-h sessions. Each session consisted of six 10-min blocks. Subjects performed a multitasking battery on a head mounted display while seated on the moving platform. The occurrence and severity of symptoms were assessed with the Motion Sickness Assessment Questionnaire (MSAQ). RESULTS: Yawning occurred predominantly in the motion condition. All yawners in motion (N = 5) were symptomatic. Compared to nonyawners (MSAQ indices: Total = 14.0, Sopite = 15.0), subjects who yawned in motion demonstrated increased severity of motion sickness and soporific symptoms (MSAQ indices: Total = 17.2, Sopite = 22.4), and reduced multitasking cognitive performance (Composite score: nonyawners = 1348; yawners = 1145). DISCUSSION: These results provide evidence that yawning may be a viable behavioral marker to recognize the onset of soporific effects and their concomitant reduction in cognitive performance.

Sopite syndrome: a revised definition.

In 1976, Graybiel and Knepton proposed the term "sopite syndrome" to describe a symptom complex centering on drowsiness and lethargy related to motion sickness. However, existing descriptions and definitions of sopite syndrome have limitations in fully conveying the appropriate information to the reader. Our objective is to propose a revised definition providing a more adequate conceptual framework for research. The proposed definition of sopite syndrome addresses the nonspecificity of soporific symptoms, the health state of the individuals, and the existence of a motion stimulus.

The H1N1 pandemic: media frames, stigmatization and coping.

BACKGROUND: Throughout history, people have soothed their fear of disease outbreaks by searching for someone to blame. Such was the case with the April 2009 H1N1 flu outbreak. Mexicans and other Latinos living in the US were quickly stigmatized by non-Latinos as carriers of the virus, partly because of news reports on the outbreak's alleged origin in Mexican pig farms. METHODS: In this exploratory study we examined the psychological processes of cue convergence and associative priming, through which many people likely conflated news of the H1N1 outbreak with pre-existing cognitive scripts that blamed Latino immigrants for a variety of social problems. We also used a transactional model of stress and coping to analyze the transcripts from five focus groups, in order to examine the ways in which a diverse collection of New England residents appraised the threat of H1N1, processed information about stereotypes and stigmas, and devised personal strategies to cope with these stressors. RESULTS: Twelve themes emerged in the final wave of coding, with most of them appearing at distinctive points in the stress and coping trajectories of focus group participants. Primary and secondary appraisals were mostly stressful or negative, with participants born in the USA reporting more stressful responses than those who were not. Latino participants reported no stressful primary appraisals, but spoke much more often than Whites or Non-Hispanic Blacks about negative secondary appraisals. When interactions between participants dealt with stigmas regarding Latinos and H1N1, Latinos in our focus groups reported using far more negative coping strategies than Whites or Non-Hispanic Blacks. When discussions did not focus on stereotypes or stigmas, Latino participants spoke much more often about positive coping strategies compared to members of these same groups. CONCLUSIONS: Participants in all five focus groups went through a similar process of stress and coping in response to the threat of H1N1, though individual responses varied by race and ethnicity. Stigmatization has often been common during pandemics, and public health and emergency preparedness practitioners can help to mitigate its impacts by developing interventions to address the social stressors that occur during outbreaks in highly-localized geographic regions.

A Strategy for Selective Deletion of Autoimmunity-Related T Cells by pMHC-Targeted Delivery.

Autoimmune diseases such as rheumatoid arthritis are caused by immune system recognition of self-proteins and subsequent production of effector T cells that recognize and attack healthy tissue. Therapies for these diseases typically utilize broad immune suppression, which can be effective, but which also come with an elevated risk of susceptibility to infection and cancer. T cell recognition of antigens is driven by binding of T cell receptors to peptides displayed on major histocompatibility complex proteins (MHCs) on the cell surface of antigen-presenting cells. Technology for recombinant production of the extracellular domains of MHC proteins and loading with peptides to produce pMHCs has provided reagents for detection of T cell populations, and with the potential for therapeutic intervention. However, production of pMHCs in large quantities remains a challenge and a translational path needs to be established. Here, we demonstrate a fusion protein strategy enabling large-scale production of pMHCs. A peptide corresponding to amino acids 259-273 of collagen II was fused to the N-terminus of the MHC_II beta chain, and the alpha and beta chains were each fused to human IgG4 Fc domains and co-expressed. A tag was incorporated to enable site-specific conjugation. The cytotoxic drug payload, MMAF, was conjugated to the pMHC and potent, peptide-specific killing of T cells that recognize the collagen pMHC was demonstrated with tetramerized pMHC-MMAF conjugates. Finally, these pMHCs were incorporated into MMAF-loaded 3DNA nanomaterials in order to provide a biocompatible platform. Loading and pMHC density were optimized, and peptide-specific T cell killing was demonstrated. These experiments highlight the potential of a pMHC fusion protein-targeted, drug-loaded nanomaterial approach for selective delivery of therapeutics to disease-relevant T cells and new treatment options for autoimmune disease.

Relation between therapy-induced changes in natriuretic peptide levels and long-term therapeutic effects on mortality in patients with heart failure and reduced ejection fraction.

AIMS: To assess whether natriuretic peptides (NPs) can be used to reliably predict long-term therapeutic effect on clinical outcomes for patients with heart failure and reduced ejection fraction (HFrEF). METHODS AND RESULTS: HFrEF intervention trials with mortality data were identified. Subsequently, we identified trials assessing therapy-induced changes in NPs. We assessed the correlation between the average short-term placebo-corrected drug or device effect on NPs and the longer-term therapeutic effect on clinical outcomes. Of 35 distinct therapies with an identifiable mortality result (n = 105 062 patients), 20 therapies had corresponding data on therapeutic effect on NPs. No correlation was observed between therapy-induced placebo-corrected change in brain natriuretic peptide or N-terminal pro-brain natriuretic peptide and therapeutic effect on all-cause mortality (ACM) (Spearman r = -0.32, P = 0.18 and r = -0.20, P = 0.47, respectively). There was no correlation between therapy-induced placebo-corrected per cent change in NP and intervention effect on ACM or ACM-heart failure hospitalizations (r = -0.30, P = 0.11 and r = 0.10, P = 0.75, respectively). CONCLUSIONS: Short-term intervention-induced changes in NP levels are not reliable predictors of therapeutic long-term effect on mortality or morbidity outcomes for patients with HFrEF.

Structural basis for the homotypic fusion of chlamydial inclusions by the SNARE-like protein IncA.

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection.

Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis.

Human PARP-1, PARP-2, and PARP-3 are key players in the cellular response to DNA damage, during which their catalytic activities are acutely stimulated through interaction with DNA strand breaks. There are also roles for these PARPs outside of the DNA damage response, most notably for PARP-1 and PARP-2 in the regulation of gene expression. Here, we describe a general method to express and purify these DNA damage-dependent PARPs from E. coli cells for use in biochemical assays and for structural and functional analysis. The procedure allows for robust production of PARP enzymes that are free of contaminant DNA that can interfere with downstream analysis. The described protocols have been updated from our earlier reported methods, most importantly to introduce PARP inhibitors in the production scheme to cope with enzyme toxicity that can compromise the yield of purified protein.

Tankyrase Sterile α Motif Domain Polymerization Is Required for Its Role in Wnt Signaling.

Tankyrase-1 (TNKS1/PARP-5a) is a poly(ADP-ribose) polymerase (PARP) enzyme that regulates multiple cellular processes creating a poly(ADP-ribose) posttranslational modification that can lead to target protein turnover. TNKS1 thereby controls protein levels of key components of signaling pathways, including Axin1, the limiting component of the destruction complex in canonical Wnt signaling that degrades ß-catenin to prevent its coactivator function in gene expression. There are limited molecular level insights into TNKS1 regulation in cell signaling pathways. TNKS1 has a sterile α motif (SAM) domain that is known to mediate polymerization, but the functional requirement for SAM polymerization has not been assessed. We have determined the crystal structure of wild-type human TNKS1 SAM domain and used structure-based mutagenesis to disrupt polymer formation and assess the consequences on TNKS1 regulation of ß-catenin-dependent transcription. Our data indicate the SAM polymer is critical for TNKS1 catalytic activity and allows TNKS1 to efficiently access cytoplasmic signaling complexes.

Much Ventured, Much Gained: Community-Engaged Data Collection by Adolescents and Young Adults.

BACKGROUND: Community-engaged data collection offers an important opportunity to build community capacity to harness the power of data and create social change. OBJECTIVES: To share lessons learned from engaging 16 adolescents and young adults from a partner community to collect data for a public opinion survey as part of a broader community-based participatory research (CBPR) project. METHODS: We conducted an analysis of archival documents, process data, and an assessment of survey assistants' experiences. LESSONS LEARNED: High-quality data were collected from a hard-to-reach population. Survey assistants benefited from exposure to research and gained professional skills. Key challenges included conducting surveys in challenging environments and managing schedule constraints during the school year. The tremendous investment made by project partners was vital for success. CONCLUSIONS: Investments required to support engaged data collection were larger than anticipated, as were the rewards, prompting greater attention to the integration of adolescents and young adults in research efforts.

Tankyrase-1 Ankyrin Repeats Form an Adaptable Binding Platform for Targets of ADP-Ribose Modification.

The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification.

Sleep Duration in Rough Sea Conditions.

INTRODUCTION: Environmental motion can affect shipboard sleep of crewmembers. Slamming and similar harsh motion may interfere with sleep, whereas mild motion and sopite syndrome may enhance sleep. If sleep needs vary by sea condition, this factor should be considered when assessing human performance at sea. The goal of this study was to assess sleep duration in different sea conditions. METHODS: Crewmembers (N = 52) from a U.S. Navy vessel participated in the study while performing their normal daily schedule of duties. Sleep was assessed with wrist-worn actigraphy. Motion sickness and sopite syndrome were assessed using standardized questionnaires. RESULTS: In rough sea conditions, crewmembers experienced increased severity of motion sickness and sopite syndrome compared to their ratings during calmer sea conditions. Crewmembers slept significantly longer during sea state 5-6 compared to sleep on days with sea state 4 (25% increase) and sea state 3-4 (30% increase). Specifically, daily sleep increased from 6.97 ± 1.24 h in sea state 3-4, to 7.23 ± 1.65 h in sea state 4, to 9.04 ± 2.90 h in sea state 5-6. DISCUSSION: Although the duration of sleep in rough seas increased significantly compared to calmer sea conditions, causal factors are inconclusive. Accumulated sleep debt, motion-induced fatigue, and sopite syndrome all may have contributed, but results suggest that motion sickness and sopite syndrome were the predominant stressors. If sleep needs increase in severe motion environments, this factor should be taken into account when developing daily activity schedules or when modeling manning requirements on modern ships.

The effect of mild motion sickness and sopite syndrome on multitasking cognitive performance.

OBJECTIVE: In this study, we investigated the effects of mild motion sickness and sopite syndrome on multitasking cognitive performance. BACKGROUND: Despite knowledge on general motion sickness, little is known about the effect of motion sickness and sopite syndrome on multitasking cognitive performance. Specifically, there is a gap in existing knowledge in the gray area of mild motion sickness. METHOD: Fifty-one healthy individuals performed a multitasking battery. Three independent groups of participants were exposed to two experimental sessions. Two groups received motion only in the first or the second session, whereas the control group did not receive motion. Measurements of motion sickness, sopite syndrome, alertness, and performance were collected during the experiment RESULTS: Only during the second session, motion sickness and sopite syndrome had a significant negative association with cognitive performance. Significant performance differences between symptomatic and asymptomatic participants in the second session were identified in composite (9.43%), memory (31.7%), and arithmetic (14.7%) task scores. The results suggest that performance retention between sessions was not affected by mild motion sickness. CONCLUSION: Multitasking cognitive performance declined even when motion sickness and soporific symptoms were mild. The results also show an order effect. We postulate that the differential effect of session on the association between symptomatology and multitasking performance may be related to the attentional resources allocated to performing the multiple tasks. Results suggest an inverse relationship between motion sickness effects on performance and the cognitive effort focused on performing a task. APPLICATION: Even mild motion sickness has potential implications for multitasking operational performance.

The communications revolution and health inequalities in the 21st century: implications for cancer control.

The radical and transformative developments in information and communication technologies (ICT) offer unprecedented opportunities to promote cancer control and enhance population and individual health. However, the current context in which these technologies are being deployed--where cancer incidence and mortality and communication are characterized by inequalities among different racial/ethnic and socioeconomic status groups--raises important questions for cancer communication research, policy, and practice. Drawing on illustrative data, this essay characterizes the communications revolution and elucidates its implications for cancer control, with a particular focus on communication inequalities and cancer disparities.

Stress training improves performance during a stressful flight.

OBJECTIVE: This study investigated whether stress training introduced during the acquisition of simulator-based flight skills enhances pilot performance during subsequent stressful flight operations in an actual aircraft. BACKGROUND: Despite knowledge that preconditions to aircraft accidents can be strongly influenced by pilot stress, little is known about the effectiveness of stress training and how it transfers to operational flight settings. METHOD: For this study, 30 participants with no flying experience were assigned at random to a stress-trained treatment group or a control group. Stress training consisted of systematic pairing of skill acquisition in a flight simulator with stress coping mechanisms in the presence of a cold pressor. Control participants received identical flight skill acquisition training but without stress training. Participants then performed a stressful flying task in a Piper Archer aircraft. RESULTS: Stress-trained research participants flew the aircraft more smoothly, as recorded by aircraft telemetry data, and generally better, as recorded by flight instructor evaluations, than did control participants. CONCLUSIONS: Introducing stress coping mechanisms during flight training improved performance in a stressful flying task. APPLICATION: The results of this study indicate that stress training during the acquisition of flight skills may serve to enhance pilot performance in stressful operational flight and, therefore, might mitigate the contribution of pilot stress to aircraft mishaps.